Compositions comprising decitabine and tetrahydrouridine and uses thereof

ABSTRACT

Compositions comprising decitabine and tetrahydrouridine for the treatment of blood disorders and hematological and solid malignancies are described.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the government, in part, via collaborationfrom the National Institutes of Health (NIH Rapid Access toInterventional Development). The internal reference number for theproject, entitled “Alternative formulations of decitabine to reactivatefetal hemoglobin expression”, was TNR42. Thus, the government hascertain rights to this invention.

BACKGROUND OF THE INVENTION

SCD is a serious congenital disease which affects 1 in 500African-Americans, as well as individuals of other racial backgrounds,exacting a substantial toll in morbidity and mortality upon theapproximately 100,000 Americans afflicted. SCD patients can havefrequent episodes of severe, debilitating pain, often requiringemergency room visits or hospitalization, and time off from work orschool. Although some patients respond to hydroxyurea (HU, the standardtreatment for symptomatic patients), and a few may be candidates forallogeneic stem cell transplantation, most patients continue to suffer.Furthermore, HU is used at doses that cause acute DNA damage andcytotoxicity, and has potential genotoxic, teratogenic andanti-fertility effects. Similarly, allogeneic stem cell transplantationis performed with cytotoxic conditioning and attendant risks oftreatment-related mortality.

A clinical research effort to develop pharmacologic inducers of HbFexpression culminated in FDA approval of the anti-metabolite HU to treatsymptomatic SCD in 1998. A recent follow-up of patients enrolled in thepivotal HU trial confirmed that a decreased risk of mortality in thesepatients correlates with HbF levels (Steinberg, M. H., et al. 2003 JAMA289:1645-1651; Rosse, W. F., et al. 2000 Am Soc Hematol Educ Program2-17). However, HbF levels are not increased in approximately 40% of HUcompliant patients (Steiberg, M. H., et al. 1997 Blood 89:1078-1088;Steinberg, M. H., et al. 1999 Expert Opin Investig Drugs 8:1823-1836;Atweh, G. F., et al. 2001 Curr Opin Hematol 8:123-130). Finally, HU isused at DNA-damaging, cytotoxic doses, potentially compounding the bonemarrow damage that accumulates in SCD.

Most cells in the body, including cancer cells or blood disorder cells,such as sickle cells, contain the same complement of genes. The functionand specialization of a cell is, therefore, determined by which of thesegenes are turned-on (activated), and which are turned-off (repressed).Activation refers to the expression of the protein encoded by the gene,while repression of the gene implies that the protein encoded by thatgene is expressed at lower levels or not at all. DNA methyl-transferase1 (DNMT1) is an enzyme which plays a critical and central role in themachinery that represses genes. Therefore, altering the levels of DNMT1within a cell can have powerful effects on the pattern ofgene-expression, function and specialization of a cell.

Decitabine (5-aza-2′-deoxycytidine) is a nucleoside analogue drug—a drugthat mimics a natural component of DNA. Decitabine is relatively uniqueamongst the large family of nucleoside analogue drugs in that it canirreversibly bind to and deplete DNMT1.

Cytidine deaminase is an enzyme that is highly expressed in the liverand intestine and rapidly destroys decitabine within the body.Tetrahydrouridine is a safe and well-tolerated pyrimidine nucleosideanalogue that inhibits cytidine deaminase. In humans, the cytidinedeaminase gene is subject to non-synonymous single nucleotidepolymorphisms which produce variants of cytidine deaminase that havedifferences in enzymatic activity of 3-fold or more (Gilbert, J. A., etal. 2006 Clin Cancer Res 12, 1794-1803; Kirch, H. C., et al. 1998 ExpHematol 26, 421-425; Yue, L., et al. 2003 Pharmacogenetics 13, 29-38).

SUMMARY OF THE INVENTION

Historically, decitabine was developed as an anti-metabolite or DNAdamaging drug intended to kill cancer cells by causing extensive damagewithin the cells. Its clinical or experimental application has not beenoptimized for the depletion of DNMT1. With an objective of usingdecitabine to deplete DNMT1 to change cell behavior, anti-metaboliteeffects that kill cells are undesirable. Optimization of decitabine todeplete DNMT1 without causing other ‘off-target’ or toxic effects isdesired.

Optimizing decitabine to deplete DNMT1 in vivo can have powerfultherapeutic benefits in a spectrum of diseases such as sickle celldisease, thalassemia, and cancers of multiple tissues. For example, insickle cell disease and β-thalassemia, by depleting DNMT1, decitabineprevents the repression of the fetal hemoglobin (HbF) gene. Theresulting increase in Hb abrogates the disease-causing effects of theabnormal sickle or thalassemia genes. Furthermore, the DNMT1 depletionby decitabine changes blood cell specialization, so that more red bloodcells are made, further addressing the debilitating anemia of theseconditions.

In cancer cells, DNMT1 depletion by decitabine prevents the repressionof differentiation genes and renews the differentiation of the cancercells—the abnormal growth of the cancer cells is caused by a block intheir normal differentiation process, which is relieved by decitabine.Of especial note, DNMT1 depletion in normal stem cells increases theirself-renewal; that is, DNMT1 depletion increases the number of normalstem cells—the opposite of its effects on cancer cells. Therefore, DNMT1depletion by decitabine could be an effective and very safe,well-tolerated cancer therapy.

Because of decitabine's unique ability to deplete DNMT1, and, therefore,alter the gene expression, function, and specialization of cells;because DNMT1 depletion by regimens of decitabine designed to depleteDNMT1 without causing DNA damage increases HbF, and produces clinicalimprovement even in sickle cell disease patients with severe illnessdespite standard of care (Saunthararajah, et al. 2008 Brit J Haematol141(1):126-9), it (decitabine) is contemplated for treatment of theseand other patients.

Because DNMT1 depletion by decitabine induces the terminaldifferentiation and apoptosis of cancer cells, while increasing theself-renewal of normal stem cells (opposite effect on cancer cellsversus normal stem cells), decitabine is additionally contemplated fortreatment of these patients.

In one embodiment of the invention, the pharmacologic objective oftherapy is to maximize time-above-threshold concentration for depletingDNMT1 (≧0.1-0.2 μM), while avoiding high peak levels (≧0.5-1 μM) thatdamage DNA.

In another embodiment of the invention, the DNMT1-depleting effectshould be intermittent. As a result, cells are allowed to divide andexhibit new behaviors. Continuous exposure to decitabine may preventcell division or even kill cells directly.

Because the currently known route of administration, regimens, andformulations of decitabine produce high peak levels of the drug, whichcan kill cells through anti-metabolite effects but produce very brieftime-above-threshold concentration for depleting DNMT1; because thecurrently known route of administration, regimens, and formulations ofdecitabine do not deplete DNMT1 intermittently to allow cell division,but, rather, produce cytotoxic or cytostatic effects; becausedestruction of decitabine by the enzyme cytidine deaminase (CDA)produces an abbreviated half-life in vivo of <20 minutes (despite an invitro half-life is 5-9 hours) (Liu, Z., et al. 2006 Rapid Common MassSpectrum 20:1117-1126); and this drastic reduction in half-life is asignificant barrier to effective in vivo translation of in vitroobservations; because pharmacogenomic variation in CDA (Gilbert, J. A.,et al. 2006 Clin Cancer Res 12, 1794-1803; Kirch, H. C., et al. 1998 ExpHematol 26, 421-425; Yue, L., et al. 2003 Pharmacogenetics 13, 29-38)produces large inter-individual variation in pharmacokinetics (PK) andclinical effects; because injections or infusions of decitabine must beadministered in the clinic or hospital, severely limiting its use insickle cell disease, where the goal is chronic disease modification forthe lifetime of the patient; because intestinal CDA-mediated destructionseverely limits its oral bioavailability (while the in vitro half-lifeof decitabine is 5-9 hrs, it has an abbreviated half-life of <20 minutesin vivo because of CDA-mediated destruction) (Liu, Z., et al. 2006 RapidCommun Mass Spectrom 20:1117-1126; Liu, Z., et al. 2007 Nucleic AcidsRes 35:e31), impeding the proposed treatment paradigm of multi-year,chronic therapy to produce sustained life-long therapeutic benefits,because malignant cells can develop resistance by destroying decitabinewith CDA (Ohta, T., et al. 2004 Oncol Rep 12:1115-1120; Hubeek, I., etal. 2005 Br J Cancer 93:1388-1394; Huang, Y., et al. 2004 Cancer Res64:4294-4301), and because malignant cells may find sanctuary fromdecitabine therapeutic effects by residing in tissues with high levelsof CDA, an oral route of administration of decitabine is contemplatedherein. Such oral administration is considered herein to decrease peaklevels and increase the time-above-threshold concentration for depletingDNMT1; to enable chronic, frequent but not daily (i.e., metronomic)therapy to sustain life-long therapeutic effects while allowing celldivision and minimizing toxicity; and to enable wide-spread use of thedrug across the globe. Additionally contemplated herein to address thelimitations and issues iterated above is the combination of decitabinewith tetrahydrouridine (THU) for oral administration.

THU inhibits CDA; THU exhibits a benign toxicity profile and awell-characterized PK; THU overcomes the intestinal and liver first-passbarriers to oral bio-availability of decitabine; THU addressespharmacogenomic variation in CDA, which produces large inter-individualvariation in decitabine PK and therapeutic effects; THU can produce amore predictable effect of a decitabine dose from individual toindividual; THU can increase the time-above-threshold concentration ofdecitabine for depleting DNMT1; THU can remove sanctuary sites formalignant cells from decitabine therapeutic effects; and THU candirectly address one mechanism of cancer cell resistance to thetherapeutic effects of decitabine. Accordingly, in one embodiment, theinvention provides a composition for oral administration comprisingabout 10 to about 150 mg/m² decitabine and about 100 to about 500 mg/m²THU.

In another embodiment, the composition is for treating a blood disorderin a subject. In yet another embodiment, the blood disorder is ahemoglobinopathy or a thalassemia. For example, the hemoglobinopathy maybe sickle cell disease.

In another embodiment, the composition is for treating hematological orsolid malignancies—cancers affecting the blood, bone marrow, and lymphnodes, including leukemia, lymphoma, and multiple myeloma, as well asrelated disorders like myelodysplastic syndrome, myeloproliferativedisease, myelofibrosis, amyloid disorders, anemia associated withmalignancy, anemia associated with inflammation including rheumatoidarthritis and inflammatory bowel diseases, anemia of chronic renalfailure, anemia associated with chronic infections such as HIV orhepatitis, anemia due to thrombocytopenia associated with malignancy,idiopathic thrombocytopenia purpura and viral diseases includingvirally-related malignancies. Examples of virally-related malignanciesinclude EBV malignancies, including, without limitation, Burkitt'slymphoma, lymphomas associated with immunosuppression, othernon-Hodgkin's lymphomas, Hodgkin's disease, nasopharyngeal carcinoma,gastric adenocarcinoma, lymphoepithelioma-like carcinomas, andimmunodeficiency-related leiomyosarcoma. In another embodiment, thecomposition is for treating cancers that affect other tissues—cancersaffecting the brain, head, neck, thyroid, bones, muscle, lung,esophagus, stomach, intestine, breasts, prostate, testes, ovaries,uterus, vagina, and skin.

In another embodiment, the invention provides a composition for oraladministration, wherein THU and decitabine are combined in a singlecapsule or tablet. For example, a composition in the form of a capsuleis contemplated comprising about 500 mg THU and about 100 mg decitabine,to facilitate an oral regimen approximating 500 mg/m² THU combined with100 mg/m² decitabine. In another embodiment of the invention,administration of the composition results in a plasma concentration ofdecitabine of 0.1-0.5 μM. In yet another embodiment, a composition ofthe invention is administered once a week or once every two weeks topatients suffering from a blood disorder. In still another embodiment, acomposition of the invention is administered between once to three timesper week to patients with cancer. In yet another embodiment, acomposition of the invention is administered between once every twoweeks to as often as three times per week in patients at risk ofdeveloping hematological or solid malignancy, or at risk of having arelapse in a previous diagnosis of hematological or solid malignancy.

In another embodiment, the invention provides a composition for oraladministration in the form of a capsule, comprising THU and decitabine,wherein the THU is released more quickly than the decitabine. Forexample, the THU might be subject to a faster dissolution rate, or theTHU might be located at the surface of the capsule, while the decitabineis located inside the capsule. In one embodiment of such a compositionof the invention, the THU is bio-available about 15 to about 180 minutesbefore the decitabine, in another, about 30 to about 60 minutes beforethe decitabine, in another, about 60 minutes before the decitabine. TheTHU may also be administered separately, in succession (with THU first,then decitabine).

In another embodiment, the composition of the invention is stored with adessicant. This could serve to extend the shelf-life of a composition ofthe invention and facilitate its distribution and use on a global scale.

In another embodiment, the invention provides a method for treating ablood disorder in a subject comprising administration of a compositioncomprising decitabine and tetrahydrouridine as described above. In yetanother embodiment, the invention provides a method for treating acancer in a subject comprising administration of a compositioncomprising decitabine and tetrahydrouridine as described above. In afurther embodiment, the administration occurs 1-3 times per week.

In one aspect, the invention provides a composition for oraladministration comprising about 10 to about 150 mg/m² decitabine andabout 100 to about 500 mg/m² tetrahydrouridine and a pharmaceuticallyacceptable excipient. In another aspect, the invention provides acomposition for oral administration comprising about 100 mg decitabineand about 500 mg tetrahydrouridine and a pharmaceutically acceptableexcipient.

In one embodiment, the composition of the invention is for treating ablood disorder in a subject. In yet another embodiment, the blooddisorder is a hemoglobinopathy or a thalassemia. In additionalembodiments, the hemoglobinopathy is a sickle cell disease, and thethalassemia is a beta thalassemia (for example, hemoglobin E betathalassemia).

In another embodiment, the composition of the invention is for treatinga hematological or solid malignancy in a subject. In yet anotherembodiment, the malignancy is selected from the group consisting ofleukemia, lymphoma, multiple myeloma, cancer of the brain, cancer of thehead, cancer of the neck, cancer of the mouth, cancer of the pharynx,cancer of the esophagus, cancer of the stomach, cancer of the intestine,cancer of the thyroid, cancer of the lungs, cancer of the mediastinum,cancer of the thymus, cancer of the mesothelium, cancer of theperitoneum, cancer of the bone, cancer of the muscle, cancer of theskin, cancer of the prostate, cancer of the breasts, cancer of theovaries, cancer of the uterus, cancer of the vagina, and virally relatedmalignancy. In an additional embodiment, the virally-related malignancyis an EBV malignancy.

In another embodiment of a composition of the invention, thetetrahydrouridine is bio-available about 15 to about 180 minutes beforethe decitabine. In another embodiment, the tetrahydrouridine isbio-available about 30 to about 60 minutes before the decitabine. In oneembodiment, the THU and decitabine are administered concurrently. Inanother embodiment, the THU is administered first, and the decitabine isadministered later.

In another aspect, the invention provides a composition for oraladministration in the form of a capsule or tablet comprising decitabineand tetrahydrouridine and a pharmaceutically acceptable excipient,wherein the tetrahydrouridine is bio-available about 15 to about 180minutes before the decitabine; or about 30 to about 60 minutes beforethe decitabine. In one embodiment, the tetrahydrouridine is located atthe surface of the capsule or tablet, and the decitabine is locatedwithin the capsule or tablet. In yet another embodiment of thecomposition of the invention, the THU is administered first in a capsuleor tablet, and the decitabine is administered later in a second capsuleor tablet.

In another aspect, the invention provides a method for treating a blooddisorder in a subject, comprising administering to the subject acomposition as described herein. In one embodiment, the blood disorderis a hemoglobinopathy or a thalassemia. In another embodiment, thesubject is provided an additional form of therapy.

In another aspect, the invention provides a method for treating ahematological or solid malignancy in a subject, comprising administeringto the subject a composition as described herein. In one embodiment, themalignancy is selected from the group consisting of leukemia, lymphoma,multiple myeloma, cancer of the brain, cancer of the head, cancer of theneck, cancer of the mouth, cancer of the pharynx, cancer of theesophagus, cancer of the stomach, cancer of the intestine, cancer of thethyroid, cancer of the lungs, cancer of the mediastinum, cancer of thethymus, cancer of the mesothelium, cancer of the peritoneum, cancer ofthe bone, cancer of the muscle, cancer of the skin, cancer of theprostate, cancer of the breasts, cancer of the ovaries, cancer of theuterus, cancer of the vagina, and virally related malignancy. In anotherembodiment, the virally related malignancy is an EBV malignancy. In yetanother embodiment, the subject is provided an additional form oftherapy.

In another aspect, the invention provides a method for decreasing theinter-individual variation in decitabine pharmacokinetics and/orclinical effects in subjects, comprising administering to the subjects acomposition as described herein.

In still another aspect, the invention provides a method for extendingthe time-above-threshold concentration for depleting DNMT1 withdecitabine in a subject and avoiding DNA-damaging high peak levels ofdecitabine, comprising administering to the subject a composition asdescribed herein.

In one embodiment of a method of the invention, the subject is human. Inanother embodiment, the method further comprises obtaining thecomposition.

Other aspects of the invention are described in or are obvious from thefollowing disclosure and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The following Detailed Description of the Invention, given by way ofExamples, but not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying figures, in which:

FIG. 1A quantifies, in bar graph form, DNA damage assessed in normalCD34+ hematopoietic cells subject to various decitabine concentrations.FIG. 1B shows histograms depicting DNA damage assessed byphosphorylation of histone H2AX. FIG. 1C graphically depicts S-phase/G2Mwith various concentrations of decitabine treatment.

FIG. 2A graphically depicts cell counts for CD34+ cells, AML1-ETO CD34+cells, and KASUMI-1 cells exposed to Ara-C vs. two different doses ofdecitabine. FIG. 2B shows, in bar graph form, colony-forming ability ofnormal CD34+ cells and AMLi-ETO CD34+ cells treated with decitabine vs.Ara-C over time.

FIG. 3A shows a table providing FAB type and cytogenic abnormalities forsamples from various patients. FIG. 3B graphically depicts the cellcounts at day 7 in untreated vs. decitabine-treated cells.

FIG. 4 graphically depicts cell counts in untreated vs.decitabine-treated cells at day 9.

FIG. 5A graphically depicts tumor volume over time in mice treated withPBS (control) vs. decitabine vs. Sunitinib vs. a combination ofdecitabine and Sunitinib. FIG. 5B graphically depicts cell number overtime for the mice.

FIG. 6 shows, in bar graph form, the repression of HoxB4, Bmi-1, andcKIT and the activation of Mcsfr, Gmcsfr, and F4/80 over time in PUERcells.

FIG. 7A graphically depicts cell proliferation over time of treatmentwith OHT vs. OHT+decitabine vs. OHT followed by decitabine. FIG. 7Bgraphically depicts macrophage differentiation (F4/80) and stem cells(c-KIT) over time of treatment with OHT vs. OHT+decitabine vs. OHTfollowed by decitabine. FIG. 7C shows, in bar graph form, Bmil, HoxB4,cKIT , F4/80, Gmcsfr, and Mcsfr over time of treatment with OHT vs.OHT+decitabine vs. OHT followed by decitabine.

FIG. 8 shows, in bar graph form, Bmil, HoxB4, cKIT, F4/80, Gmcsfr, andMcsfr over time in PUERshRunx1 cells.

FIG. 9A shows, in bar graph form, PU.1, CEBPα, CEBPε, and GATA-1 invarious patient samples. FIG. 9B lists, in table form, the WHOclassification and cytogenic abnormalities for each of the samples. FIG.9C shows, in bar graph form, precursor gene methylation anddifferentiation gene methylation for normal CD34+ cells, normal bonemarrow cells, MDS cells, and AML cells.

FIG. 10 plots the survival of leukemic mice untreated vs. treated withdecitabine.

FIG. 11 graphically depicts decitabine peak levels andtime-above-threshold for DNMT1 depletion produced by subcutaneous vs.oral decitabine.

FIGS. 12A and 12B show, in bar graph form, DNMT1 depletion indecitabine-sensitive and decitabine-resistant cell lines (in terms of %control growth for the bar graphs).

FIG. 13 graphically depicts HbF expression elevation over timeassociated with decitabine.

FIG. 14 graphically depicts plasma concentration-time curves ofdecitabine in non-human primates.

FIG. 15 graphically depicts plasma concentration-time curves ofdecitabine following oral administration to baboons at 10 mg/kg.

FIG. 16 graphically depicts plasma concentration-time curves ofdecitabine in baboons following oral administration alone or 60 minutesafter THU.

FIG. 17 graphically depicts plasma concentration-time curves ofdecitabine in baboons following oral administration alone at 5 mg/kg or60 minutes after 2 or 20 mg/kg THU.

FIG. 18A depicts, in bar graph form, the distribution of AUC in 7animals treated with decitabine 10 mg/kg alone by oral gavage or THU 20mg/kg by oral gavage followed by decitabine 5 mg/kg by oral gavage 60minutes later. Horizontal line in box-plot=median, boxboundaries=interquartile range, connecting diagonal line joins the meanin the two groups. The wide separation of median and mean in thedecitabine only group is narrowed substantially in the decitabine-THUgroup. The difference in medians between the two groups was notstatistically significant (p=0.22, Wilcoxon). The difference in meansbetween the two groups was not statistically significant (p=0.08, pairedt-test). FIG. 18B depicts, in bar graph form, the results of A brokendown by individual animals.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “treating”, as used herein, refers to altering the diseasecourse of the subject being treated. Therapeutic effects of treatmentinclude, without limitation, preventing occurrence or recurrence ofdisease, alleviation of symptom(s), diminishment of direct or indirectpathological consequences of the disease, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

Hematological malignancies are a group of neoplasms that arise throughmalignant transformation of bone marrow derived cells. They can besubdivided into myeloid and lymphoid disorders and include, withoutlimitation, acute lymphoblastic leukemia, chronic lymphoid leukemia,diffuse large B-cell lymphoma, follicular centre lymphoma, Hodgkinslymphoma, mantle cell lymphoma, marginal zone lymphoma, Waldenstrom'smacroglobulinemia, myeloma, monoclonal gammopathy of uncertainsignificance, large granular lymphocyte syndrome, T-prolymphocyticsyndrome, Sezary syndrome, lymphoma, angio-immunoblastic lymphoma,anaplastic large cell lymphoma, mycosis fungoides, lymphomatoidpapulosis, small intestinal lymphoma, acute myeloid leukemia,myelodysplastic syndrome, myeloproliferative disorders, mylofibrosis,paroxysmal nocturnal hemoglobinuria, aplastic anemia, anemia associatedwith malignancy, thrombocytopenia associated with malignancy,virally-related malignancies, post-transplant lymphoproliferativesyndrome, NK/T lymphoma, AIDS-related lymphoma, Burkitt's lymphoma, andnon-Burkitt's small cell lymphoma. Chronic lymphocytic leukemia,non-Hodgkin lymphoma, and myeloid leukemia are particularly prevalentmalignancies contemplated for treatment herein.

Solid tumor malignancies are a group of neoplasms that arise through themalignant transformation of cells in non-blood-related tissues. Theseinclude, without limitation, malignancies of the brain, head, neck,mouth, pharynx, esophagus, stomach, intestine, thyroid, lungs,mediastinum, thymus, mesothelium, peritoneum, bone, muscle, skin,prostate, breasts, ovaries, uterus, and vagina.

Hemoglobinopathies and thalassemias can both be characterized as “blooddisorders” and are caused by abnormalities in the globin genes. Blooddisorders include disorders that can be treated, prevented, or otherwiseameliorated by the administration of a compound of the invention. Ablood disorder is any disorder of the blood and blood-forming organs.The term blood disorder includes nutritional anemias (e.g., irondeficiency anemia, sideropenic dysphasia, Plummer-Vinson syndrome,vitamin B12 deficiency anemia, vitamin B12 deficiency anemia due tointrinsic factor, pernicious anemia, folate deficiency anemia, and othernutritional anemias), myelodysplastic syndrome, bone marrow failure oranemia resulting from chemotherapy, radiation or other agents ortherapies, hemolytic anemias (e.g., anemia due to enzyme disorders,anemia due to phosphate dehydrogenase (G6PD) deficiency, favism, anemiadue to disorders of glutathione metabolism, anemia due to disorders ofglycolytic enzymes, anemias due to disorders of nucleotide metabolismand anemias due to unspecified enzyme disorder), thalassemia,α-thalassemia, β-thalassemia (for example, hemoglobin E betathalassemia), δβ-thalassemia, thalassemia trait, hereditary persistenceof fetal hemoglobin (HPFP), and other thalassemias, sickle celldisorders (sickle cell anemia with crisis, sickle cell anemia withoutcrisis, double heterozygous sickling disorders, sickle cell trait andother sickle cell disorders), hereditary hemolytic anemias (hereditaryspherocytosis, hereditary elliptocytosis, other hemaglobinopathies andother specified hereditary hemolytic anemias, such as stomatocyclosis),acquired hemolytic anemia (e.g., drug-induced autoimmune hemolyticanemia, other autoimmune hemolytic anemias, such as warm autoimmunehemolytic anemia, drug-induced non-autoimmune hemolytic anemia,hemolytic-uremic syndrome, and other non-autoimmune hemolytic anemias,such as microangiopathic hemolytic anemia); aplastic anemias (e.g.,acquired pure red cell aplasia (erythoblastopenia), other aplasticanemias, such as constitutional aplastic anemia and fanconi anemia,acute post-hemorrhagic anemic, and anemias in chronic diseases),coagulation defects (e.g., disseminated intravascular coagulation(difibrination syndrome)), hereditary factor VIII deficiency (hemophiliaA), hereditary factor IX deficiency (Christmas disease), and othercoagulation defects such as Von Willebrand's disease, hereditary factorXi deficiency (hemophilia C), purpura (e.g., qualitative plateletdefects and Glanzmann's disease), neutropenia, agranulocytosis,functional disorders of polymorphonuclear neutrophils, other disordersof white blood cells (e.g., eosinophilia, leukocytosis, lymophocytosis,lymphopenia, monocytosis, and plasmacyclosis), diseases of the spleen,methemoglobinemia, other diseases of blood and blood forming organs(e.g., familial erythrocytosis, secondary polycythemia, essentialthrombocytosis and basophilia), thrombocytopenia, infectious anemia,hypoproliferative or hypoplastic anemias, hemoglobin C, D and E disease,hemoglobin lepore disease, and HbH and HbS diseases, anemias due toblood loss, radiation therapy or chemotherapy, or thrombocytopenias andneutropenias due to radiation therapy or chemotherapy, sideroblasticanemias, myelophthisic anemias, antibody-mediated anemias, and certaindiseases involving lymphoreticular tissue and reticulohistiocytic system(e.g., Langerhans' cell hystiocytosis, eosinophilic granuloma,Hand-Schuller-Christian disease, hemophagocytic lymphohistiocytosis, andinfection-associated hemophagocytic syndrome).

The thalassemias are classified according to which chain of thehemoglobin molecule is affected. In α thalassemias, production of the αglobin chain is affected, while in β thalassemia, production of the βglobin chain is affected. β globin chains are encoded by a single geneon chromosome 11.

Beta thalassemias are due to mutations in the HBB gene on chromosome 11.The severity of the disease depends on the nature of the mutation.Mutations are characterized as (β° or β thalassemia major) if theyprevent any formation of β chains (which is the most severe form of betathalassemia); they are characterized as (β⁺or β thalassemia intermedia)if they allow some β chain formation to occur. In either case, there isa relative excess of a chains, but these do not form tetramers: rather,they bind to the red blood cell membranes, producing membrane damage,and at high concentrations they form toxic aggregates.

The term “pharmaceutically acceptable excipient”, as used herein, refersto carriers and vehicles that are compatible with the active ingredient(for example, a compound of the invention) of a pharmaceuticalcomposition of the invention (and preferably capable of stabilizing it)and not deleterious to the subject to be treated. For example,solubilizing agents that form specific, more soluble complexes with thecompounds of the invention can be utilized as pharmaceutical excipientsfor delivery of the compounds. Suitable carriers and vehicles are knownto those of extraordinary skill in the art. The term “excipient” as usedherein will encompass all such carriers, adjuvants, diluents, solvents,or other inactive additives. Suitable pharmaceutically acceptableexcipients include, but are not limited to, water, salt solutions,alcohol, vegetable oils, polyethylene glycols, gelatin, lactose,amylose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid monoglycerides and diglycerides, petroethralfatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc.The pharmaceutical compositions of the invention can also be sterilizedand, if desired, mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like, which do not deleteriously react withthe active compounds of the invention.

The term “bio-available”, as referred to herein, refers to when theactive agent (tetrahydrouridine or decitabine) can be absorbed and usedby the body. “Orally bio-available” indicates that the agent has beentaken by mouth and can be absorbed and used by the body.

The term “subject” as used herein refers to a vertebrate, preferably amammal, more preferably a primate, still more preferably a human.Mammals include, without limitation, humans, primates, wild animals,feral animals, farm animals, sports animals, and pets.

The term “obtaining” as in “obtaining the composition” is intended toinclude purchasing, synthesizing, or otherwise acquiring the composition(or agent(s) of the composition).

The terms “comprises”, “comprising”, are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like.

The invention can be understood more fully by reference to the followingdetailed description and illustrative examples, which are intended toexemplify non-limiting embodiments of the invention.

II. Additional Embodiments of the Invention

Pharmaceutical Compositions

In one embodiment, pharmaceutical compositions and dosage forms of theinvention comprise about 10 to about 150 mg/m² decitabine and about 100to about 500 mg/m² THU and a pharmaceutically acceptable excipient, inrelative amounts and formulated in such a way that a givenpharmaceutical composition or dosage form reactivates fetal hemoglobin(HbF) expression and/or expands normal hematopoietic stem cells and/orcauses a shift to the erythropoietic lineage. In another embodiment, thepharmaceutical compositions and dosage forms of the invention compriseabout 100 mg decitabine and about 500 mg THU and a pharmaceuticallyacceptable excipient.

In another embodiment, the compositions of the invention are formulatedin such a way that a given pharmaceutical composition or dosage formdecreases the aberrant repression of differentiation-related genes inhematological or solid malignancies, thus reducing or inhibiting thegrowth of transformed (cancer) cells. In another embodiment of theinvention, such pharmaceutical compositions and dosage forms compriseone or more additional active agents. For the treatment of hematologicalor solid malignancies, such additional active agents includechemotherapeutic agents known in the art.

The compositions of the invention are administered orally in effectivedosages, depending upon the weight, body surface area, and condition ofthe subject being treated. Variations may occur depending upon thespecies of the subject being treated and its individual response to saidmedicament, as well as on the type of pharmaceutical formulation chosenand the time period and interval at which such administration is carriedout.

In one embodiment, the pharmaceutical compositions of the invention maybe administered alone or in combination with other known compositionsfor treating blood disorders in a subject, e.g., a mammal Preferredmammals include cats, dogs, pigs, rats, mice, monkeys, chimpanzees,baboons and humans. In one embodiment, the subject is suffering from ablood disorder. In another embodiment, the subject is at risk ofsuffering from a blood disorder.

In another embodiment, the pharmaceutical compositions of the inventionmay be administered alone or in combination with other knowncompositions for treating hematological malignancies in a subject, e.g.,a mammal Preferred mammals include cats, dogs, pigs, rats, mice,monkeys, chimpanzees, baboons and humans. In one embodiment, the subjectis suffering from a hematological malignancy. In another embodiment, thesubject is at risk of suffering from a hematological malignancy.

The language “in combination with” a known composition is intended toinclude simultaneous administration of the composition of the inventionand the known composition, administration of the composition of theinvention first, followed by the known composition and administration ofthe known composition first, followed by the composition of theinvention. Any of the composition known in the art for treating blooddisorders or hematological malignancies can be used in the methods ofthe invention.

The administration of the compositions of the invention may be carriedout in single or multiple doses. For example, the novel compositions ofthis invention can be administered advantageously in a wide variety ofdifferent dosage forms, i.e., they may be combined with variouspharmaceutically acceptable inert carriers in the form of tablets,dragees, capsules, lozenges, troches, hard candies, aqueous suspensions,elixirs, syrups, and the like. Such carriers include solid diluents orfillers, sterile aqueous media and various non-toxic organic solvents,etc. Moreover, oral pharmaceutical compositions can be suitablysweetened and/or flavored. In general, the therapeutically-effectivecompounds of this invention are present in such dosage forms atconcentration levels ranging from about 5.0% to about 70% by weight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch (and preferably corn, potato or tapioca starch), alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

Sustained release compositions can be formulated including those whereinthe active component is derivatized with differentially degradablecoatings, e.g., by microencapsulation, multiple coatings, etc. In oneembodiment of such a composition of the invention, the THU isbio-available about 15 to about 180 minutes before the decitabine. Inanother embodiment, the THU is bio-available about 30 to about 60minutes before the decitabine.

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the particularcompositions formulated. Optimal administration rates for a givenprotocol of administration can be readily ascertained by those skilledin the art using conventional dosage determination tests conducted withregard to the foregoing guidelines.

It will also be understood that normal, conventionally known precautionswill be taken regarding the administration of the compounds of theinvention generally to ensure their efficacy under normal usecircumstances. Especially when employed for therapeutic treatment ofhumans and animals in vivo, the practitioner should take all sensibleprecautions to avoid conventionally known contradictions and toxiceffects.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. This aspect of the invention willbe readily apparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences (1990) 18th ed., Mack Publishing, Eastern Pa.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichthe compound of the invention will decompose. Such compounds, which arereferred to herein as “stabilizer” include, but are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

The interrelationship of dosages for animals and humans (based onmilligrams per meter squared of body surface) is described in Freireich,et al. 1966 Cancer Chemother Rep 50: 219. Body surface area may beapproximately determined from height and weight of the patient. See,e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y., 1970, 537.

Methods of Treatment

In one embodiment of the invention, a composition of the invention isadministered to a patient in need of treatment of a blood disorder. Inanother embodiment of the invention, a composition of the invention isadministered to a patient in need of treatment of a hematological orsolid malignancy. Other conditions, diseases and disorders that wouldbenefit from such uses are known to those of skill in the art.

Responsiveness of the disease to compositions of the invention can bemeasured directly by comparison against conventional drugs (for example,for hematological or solid malignancies, chemotherapeutics; for certainblood disorders, hydroxyurea, histone deacetylase inhibitors, orerythropoietin), or can be inferred based on an understanding of diseaseetiology and progression. For example, there are a number of HbFexpression assay systems that are widely accepted in the art aspredictive of in vivo effects. Thus, the showing that a compound of thisinvention induces HbF expression in these assays is evidence of theclinical utility of these for treating a hemoglobinopathy and/or athalassemia, i.e., a blood disorder.

In one embodiment of the invention, “treatment” or “treating” refers toan amelioration of a hemoglobinopathy and/or a thalassemia, i.e., ablood disorder, or at least one discernible symptom thereof. In anotherembodiment, “treatment” or “treating” refers to an amelioration of atleast one measurable physical parameter, not necessarily discernible bythe patient. In yet another embodiment, “treatment” or “treating” refersto inhibiting the progression of a hemoglobinopathy and/or athalassemia, i.e., a blood disorder, either physically, e.g.,stabilization of a discernible symptom, physiologically, e.g.,stabilization of a physical parameter, or both. In yet anotherembodiment, “treatment” or “treating” refers to delaying the onset of ahemoglobinopathy and/or a thalassemia, i.e., a blood disorder, orsymptoms thereof.

In another embodiment of the invention, “treatment” or “treating” refersto an amelioration of a hematological or solid malignancy or at leastone discernible symptom thereof. In another embodiment, “treatment” or“treating” refers to an amelioration of at least one measurable physicalparameter, not necessarily discernible by the patient. In yet anotherembodiment, “treatment” or “treating” refers to inhibiting theprogression of cancer, either physically, e.g., stabilization of adiscernible symptom, physiologically, e.g., stabilization of a physicalparameter, or both. In yet another embodiment, “treatment” or “treating”refers to delaying the onset of a hematological or solid malignancy orsymptoms thereof.

The compositions of the invention can be assayed in vitro or in vivo,for the desired therapeutic or prophylactic activity, prior to use inhumans. For example, animal model systems can be used to demonstrate thesafety and efficacy of compounds of this invention.

Without wishing to be bound by theory, it is believed that thecompositions of this invention induce gene expression, for example,fetal hemoglobin expression and, as a result, may be used to treat orprevent a hemoglobinopathy and/or a thalassemia, i.e., a blood disorder.Further without wishing to be bound by theory, it is believed that thecompositions of this invention bind to and deplete DNA methyltransferase(specifically, DNMT1), decreasing repression or aberrant repression ofgenes that could have therapeutic effects if they were expressed, and,as a result, may be used to treat or prevent hematological or solidmalignancies. It should be noted, however, that the compositions mightact by a secondary or a different activity, such as, without limitation,stimulating hematopoiesis, erythropoiesis, and increasing self-renewalof normal stem cells.

The altered expression of genes could also increase recognition ofmalignant cells by cells of the immune system, whether that immunesystem is the patient's own or an allogeneic immune system reconstitutedthrough allogeneic stem cell transplantation or infusion of donorlymphocytes.

Combination Therapy

The herein-described methods for treating a hemoglobinopathy and/or athalassemia, i.e., a blood disorder, in a subject can further compriseadministering to the subject being administered a composition of thisinvention, an effective amount of one or more other therapeutic agents.In one embodiment of the invention where another therapeutic agent isadministered to a subject, the effective amount of the composition ofthe invention is less than its effective amount would be where the othertherapeutic agent is not administered. In another embodiment, theeffective amount of the other therapeutic agent is less than itseffective amount would be where the composition of the invention is notadministered.

The herein-described methods for treating a hematological or solidmalignancy in a subject can further comprise administering to thesubject being administered a composition of this invention, an effectiveamount of one or more other therapeutic agents. In one embodiment of theinvention where another therapeutic agent is administered to a subject,the effective amount of the composition of the invention is less thanits effective amount would be where the other therapeutic agent is notadministered. In another embodiment, the effective amount of the othertherapeutic agent is less than its effective amount would be where thecomposition of the invention is not administered.

In some aspects described herein, the method includes an additionaltherapeutic modality. For example, the additional therapeutic modalityis radiation therapy or a cytotoxic chemotherapy agent, such as ananti-metabolite (e.g., 5-FU, with leucovorin), irinotecan, (or othertopoisomerase inhibitor), doxorubicin, HDAC inhibitors, anti-viralagents, anti-retroviral agents, or any combination all of these agents,including administration of all of these agents. Included withanti-viral agent treatment may be pre-treatment with an agent thatinduces the expression of viral thymidine kinase.

In additional aspects described herein, the methods can includemonitoring the subject for the pharmacodynamic effect of therapy, e.g.,for depletion of DNMT1 in normal and malignant cells.

The methods can further include the step of monitoring the subject,e.g., for a reduction in one or more of: a reduction in tumor size;reduction in cancer markers, e.g., levels of cancer specific antigen;reduction in the appearance of new lesions, e.g., in a bone scan; areduction in the appearance of new disease-related symptoms; ordecreased or stabilization of size of soft tissue mass; or any parameterrelated to improvement in clinical outcome. The subject can be monitoredin one or more of the following periods: prior to beginning oftreatment; during the treatment; or after one or more elements of thetreatment have been administered. Monitoring can be used to evaluate theneed for further treatment with the composition of the invention or foradditional treatment with additional agents. Generally, a decrease in orstabilization of one or more of the parameters described above isindicative of the improved condition of the subject. Information aboutthe monitoring can be recorded, e.g., in electronic or digital form.

The treatment methods disclosed herein can be used in combination withone or more additional treatment modalities, including, but not limitedto: surgery; radiation therapy, and chemotherapy.

With reference to the methods disclosed herein, the term “combination”refers to the use of one or more additional agents or therapies to treatthe same patient, wherein the use or action of the agents or therapiesoverlap in time. The additional agents or therapies can be administeredat the same time as the composition of the invention is administered, orsequentially in any order. Sequential administrations areadministrations that are given at different times. The time betweenadministration of the one agent and another agent can be minutes, hours,days, or weeks.

The additional agent or therapy can also be another anti-cancer agent ortherapy. Non-limiting examples of anti-cancer agents include, e.g.,anti-microtubule agents, topoisomerase inhibitors, antimetabolites,mitotic inhibitors, alkylating agents, intercalating agents, agentscapable of interfering with a signal transduction pathway, agents thatpromote apoptosis, radiation, and antibodies against othertumor-associated antigens (including naked antibodies, immunotoxins andradioconjugates). Examples of the particular classes of anti-canceragents are provided in detail as follows: antitubulin/antimicrotubule,e.g., paclitaxel, vincristine, vinblastine, vindesine, vinorelbin,taxotere; topoisomerase I inhibitors, e.g., irinotecan, topotecan,camptothecin, doxorubicin, etoposide, mitoxantrone, daunorubicin,idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantronehydrochloride; antimetabolites, e.g., 5-fluorouracil (5-FU),methotrexate, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,cytarabine/Ara-C, trimetrexate, gemcitabine, acivicin, alanosine,pyrazofurin, N-Phosphoracetyl-L-Asparate=PALA, pentostatin,5-azacitidine, 5-Aza 2′-deoxycytidine, ara-A, cladribine,5-fluorouridine, FUDR, tiazofurin,N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenyl]-L-glutamicacid; alkylating agents, e.g., cisplatin, carboplatin, mitomycin C, BCNU(Carmustine), melphalan, thiotepa, busulfan, chlorambucil, plicamycin,dacarbazine, ifosfamide phosphate, cyclophosphamide, nitrogen mustard,uracil mustard, pipobroman, 4-ipomeanol; agents acting via othermechanisms of action, e.g., dihydrolenperone, spiromustine, anddesipeptide; biological response modifiers, e.g., to enhance anti-tumorresponses, such as interferon; apoptotic agents, such as actinomycin D;and anti-hormones, for example anti-estrogens such as tamoxifen or, forexample antiandrogens such as4′-cyano-3-(4-fluorophenylsulphonyl)-2-hydroxy-2-methyl-3′-(trifluoromethyl)propionanilide.

Histone deacetylase inhibitors (HDAC inhibitors), a class of compoundsthat interfere with the function of histone deacetylase, are likewisecontemplated as an additional agent for combination therapy. HDACinhibitors include, without limitation, hyroxamic acids (for example,Trichostatin A), cyclic tetrapeptides (for example, trapoxin B),depsipeptides (for example, romidepsin), benzamides, electrophilicketones, aliphatic acid compounds (for example, phenylbutyrate, valproicacid), SAHA/Vorinostat, FK228, Belinostat/PXD101, Panobinostat, MS-275,LAQ824/LBH589, CI994, MGCD0103, nicotinamide, NAD derivatives,dihydrocoumarin, naphthopyranone, 2-hydroxynaphthaldehydes,dicarboxamide derivatives, pyridyl and pyrimidinyl derivatives,4-carboxybenzylamino derivatives, fluorinated arylamide derivatives,stilbene-like compounds, 3-(4-amidopyrrol-2-ylmethlidene)-2-indolinonederivatives and phenoxazinone.

A combination therapy can include administering an agent that reducesthe side effects of other therapies. The agent can be an agent thatreduces the side effects of anti-cancer treatments. A combinationaltherapy can also include administering an agent that reduces thefrequency of administration of other therapies. The agent can be anagent that decreases growth of tumor after the anti-cancer effects ofother therapies have decreased.

Useful combination therapies will be understood and appreciated by thoseof skill in the art. Potential advantages of such combination therapiesinclude the ability to use less of each of the individual activeingredients to minimize toxic side effects, synergistic improvements inefficacy, improved ease of administration or use, and/or reduced overallexpense of compound preparation or formulation. For example, thecompounds of the invention may be administered to the subject fortreatment of a hemoglobinopathy and/or a thalassemia, i.e., a blooddisorder, in combination with one or more cytokines. In one embodiment,the cytokine is selected from the group consisting of IL-3, GM-CSF,G-CSF, stem cell factor (SCF) and IL-6.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features. From the above description and the examples thatfollow, one skilled in the art can easily ascertain the essentialcharacteristics of the present invention, and without departing from thespirit and scope thereof, can make various changes and modifications ofthe invention to adapt it to various usages and conditions. For example,the compounds of the invention may be used as research tools (forexample, to isolate new targets for performing drug discovery). Thecompounds may, for instance, be radiolabelled for imaging tissue ororgans or be used to form bioconjugates for affinity assays. These andother uses and embodiments of the compounds and compositions of thisinvention will be apparent to those of ordinary skill in the art.

The disclosure also encompasses all possible permutations of the claimset, as if they were multiple dependent claims.

Various embodiments of the disclosure could also include permutations ofthe various elements recited in the claims as if each dependent claimwas multiple dependent claim incorporating the limitations of each ofthe preceding dependent claims as well as the independent claims. Suchpermutations are expressly within the scope of this disclosure.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims. All references cited herein are incorporated in theirentirety by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of the present invention and are covered by thefollowing claims. The contents of all references, patents, and patentapplications cited throughout this application are hereby incorporatedby reference. The appropriate components, processes, and methods ofthose patents, applications and other documents may be selected for thepresent invention and embodiments thereof.

The description of the present invention has been presented for purposesof illustration and description, but is not intended to be exhaustive orlimiting of the invention to the form disclosed. The scope of thepresent invention is limited only by the scope of the following claims.Many modifications and variations will be apparent to those of ordinaryskill in the art. The embodiment described and shown in the figures waschosen and described in order to best explain the principles of theinvention, the practical application, and to enable others of ordinaryskill in the art to understand the invention for various embodimentswith various modifications as are suited to the particular usecontemplated.

The invention is further defined by reference to the following examplesdescribing in detail the preparation of compounds of, the invention. Itwill be apparent to those skilled in the art that many modifications,both to materials and methods, may be practiced without departing fromthe purpose and interest of this invention. The following examples areset forth to assist in understanding the invention and should not beconstrued as specifically limiting the invention described and claimedherein. Such variations of the invention, including the substitution ofall equivalents now known or later developed, which would be within thepurview of those skilled in the art, and changes in formulation or minorchanges in experimental design, are to be considered to fall within thescope of the invention incorporated herein.

While the invention has been particularly shown and described withreference to a number of embodiments, it would be understood by thoseskilled in the art that changes in the form and details may be made tothe various embodiments disclosed herein without departing from thespirit and scope of the invention and that the various embodimentsdisclosed herein are not intended to act as limitations on the scope ofthe claims.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Decitabine Can Deplete DNMT1 in Normal Hematopoietic Stem andProgenitor Cells Without Causing Measurable DNA Damage or Apoptosis

Decitabine is shown herein to deplete DNMT1 without causing measurableDNA damage. Decitabine at a concentration of 0.2-0.5 μM depletes DNMT1in normal hematopoietic precursor cells (data not shown). Normal CD34+hematopoietic precursor cells were isolated from cord-blood. A Q-dotbased immunofluorescence assay quantifies DNMT1 depletion by variousdoses of decitabine. Q-dot conjugated 2° ab against anti-DNMT1 1° aballows quantification of DNMT1 depletion in normal human CD34+hematopoietic cells exposed to various levels of decitabine. DNMT1 wasquantified in 500 cells for each treatment condition. DAPI was used tostain the nuclei Image-quant software is used to quantify the DNMT1 bythe Mean Intensity Fluorescence (MIF) variable. Decitabine levels wereassessed in μM.

FIGS. 1A and B) Decitabine at a concentration of 0.5 μM, added to normalhematopoietic stem and progenitor cells 3×/wk, does not cause measurableDNA damage. However, decitabine 1.0 μM causes measurable DNA damage. A)The normal CD34+ hematopoietic cells subject to the decitabineconcentrations above were assessed for DNA damage by the FastMicro-method for measuring DNA scission 24 hrs after DAC exposure(Riccardi, R., et al. 1982 Cancer Res 42:1736-1739). In contrast,equimolar doses of Ara-C (Ara-C 0.5 μM) and clinically relevant levelsof hydroxyurea (HU 500 μM), cause significant DNA damage (decitabinedose 0.5 μM D1, 3, 5; Ara-C dose 0.5 μM D 1, 3, 5; hydroxyurea dose 500μM D1-5). Measurement was performed on D6. B) DNA damage was alsoassessed by phosphorylation (γ) of histone H2AX, an early marker of DNArepair again demonstrating the above concentrations of Ara-C (Ara-C) andclinically relevant levels of HU cause significant DNA damage, but notequimolar amounts of decitabine.

Dark histogram=isotype control for flow-cytometric analysis. Lighthistograms show the γH2AX staining for untreated control cells.Measurement was performed on D6.

FIG. 1C) DAC, at these non-DNA-damaging but DNMT1-depleting levels,produces transient cell-cycle arrest followed by reboundhyperproliferation. Cell cycle status was measured at the varioustimepoints by flow-cytometric assessment of propidium iodide staining.Decitabine treatment causes cytostasis with a rebound increase inS-phase/G2M that occurs approximately 48 h after drug exposure. Resultsare expressed as a percentage of untreated control.

The kinetics of DNMT1 depletion and recovery in normal hematopoieticprecursors exposed to a 1× addition of DAC 0.5 μM. DNMT1 was quantifiedwith a Q dot-based assay. Nuclei were stained with DAPI. Decitabine wasfound to cause minimal or no evidence of apoptosis by flow-cytometricmeasurement of annexin V-FITC and 7AAD double staining (data not shown).Equimolar doses of Ara-C (Ara-C) and clinically relevant levels of HU(Kreis, W., et al. 1991 Leukemia 5:991-998) cause significant apoptosisand cell-death. Measurement was performed on D6.

Therefore, the cytotoxic effects of decitabine can be separated from itsDNMT1 depleting effects at concentrations between 0.2-0.5 μM. Therefore,for non-cytotoxic epigenetic therapy, the pharmacologic goal is toincrease time above threshold concentration required to deplete DNMT(approximately 0.2 μM) while avoiding high peak levels that cause DNAdamage (>0.5-1.0 μM).

Example 2 DNMT1-Depleting But Non-DNA Damaging, Doses of Decitabine HaveOpposite Effects on Normal Stem-Cells (HSC) Versus Leukemia Cells

CD34 cells transduced with AML1-ETO recapitulated some features ofleukemia stem-cells (LSC) (impaired differentiation and increasedself-renewal) and represented a first-hit or early model of leukemictransformation. The Kasumi-1 cell-line is derived from a patient withAML1-ETO leukemia and represents a late-stage model of transformationand malignant evolution. Both of these models terminally differentiatewith non-DNA-damaging but DNMT1-depleting doses of decitabine. FIG. 2A)Cell counts: an ideal therapeutic index was seen, with proliferation ofnormal cells while leukemia cells decline. In contrast, standard therapy(Ara-C), given at equimolar doses was more devastating to normal cells.Normal cells retained primitive morphology with decitabine treatment(data not shown). In contrast, CD34 AML1-ETO and Kasumi-1 cellsmorphologically differentiated (decreased nuclear cytoplasmic ratio,nuclear segmentation or condensation, cytoplasmic granulation andvacuolation). FIG. 2B) Normal CD34+ cells treated with decitabinedemonstrated increased self-renewal (Milhem, M., et al. 2004 Blood103:4102-4110), therefore, decitabine treatment maintains colony-formingability. In contrast, CD34 AML1-ETO cells (leukemia cells) terminallydifferentiate with decitabine treatment and colony forming ability isabrogated (data not shown).

Example 3 DNMT1 Depletion Without DNA Damage Produces TerminalDifferentiation (Not Immediate Apoptosis) of Primary Leukemia Cells FromPatients

Primary leukemia cells obtained from bone marrow or peripheral blood(with informed consent on an IRB approved protocol) were cultured inmedia supplemented with cytokines with or without decitabine 0.5 μMadded 2×/week. These concentrations of decitabine did not cause DNAdamage or immediate apoptosis. FIG. 3A) The samples were obtained from aspectrum of AML sub-types. FIG. 3B) Cell counts at D7 in untreatedcontrol versus decitabine-treated cells. Giemsa staining demonstratedterminal myelomonocytic differentiation of decitabine treated cells in12 of 14 cases (data not shown). In cases of resistance, DNMT1expression in the cells was retained, indicating failure of decitabineactivity, rather than resistance to the effects of DNMT1 depletion (datanot shown).

Decitabine 0.5 μM, concentrations that did not cause early apoptosis asmeasured by Annexin staining, terminally differentiated solid tumorcell-lines (renal cancer, small cell lung cancer, hepatocellular cancer,prostate cancer, bladder cancer), producing morphologic changes ofdifferentiation (increased cell-size, decreased nuclear-cytoplasmicratio). Melanoma is a cancer that is resistant to conventionalapoptosis-based therapy. Decitabine 0.5 μM induced changes of terminaldifferentiation in 7 melanoma cell lines. FIG. 4) Cell counts in controland decitabine treated cells at D9. Giemsa-stained control and treatedcells at D8—morphologic changes indicated that decitabine treatmentinduced differentiation (data not shown).

Example 4 A Differentiation Therapy Regimen of Decitabine is EffectiveAnd Very Well Tolerated in a Xeno-Transplant Model of Avastin-ResistantRenal Cancer

In an effort to apply the proposed formulation and regimen to treatdifferent cancer histologies, a renal cell cancer cell-line (Reno-1) wasdeveloped from a surgical sample of renal cancer and used in axeno-transplantation experiment. In a xenograft model of resistant renalcancer, SQ decitabine given 3×/week at a dose of 1 mg/m2 (starting onDay 9—tumor vol. 100) significantly decreased tumor volume withoutevidence of toxicity in the mice (no change in weight, appearance orblood counts). This tumor was relatively resistant to avastin andsunitinib, standard agents used to treat renal cancer. FIG. 5A)Decitabine (DAC) decreased tumor volume (p<0.001, t-test), compared tocontrol (PBS) or Sunitinib. Sunitinib antagonized the effect ofdecitabine, presumably by cytostasis that decreased the S-phasedependent incorporation of decitabine into tumor. H&E staining ofparaffin embedded sections demonstrated necrosis in thedecitabine-treated samples (data not shown). The percentage of necrosisin each tissue section was evaluated in a blinded fashion. ThePBS-treated animals (controls) had dense, healthy tissue which is poorlystaining (grey tissue highlighted with white arrows). FIG. 5B)Paraffin-embedded sections do not lend themselves to analysis forcytological detail. Therefore, the morphology of Giemsa-stained Reno-1cells cultured with decitabine was examined in vitro. Decitabine-treatedcells increase in size and demonstrate prominent nuclear chromatinclumps.

Example 5 The PUER Model of Pu.1-Mediated Hematopoietic DifferentiationProvides an Insight Into the Mechanisms That Underlie the OppositeEffects of DNMT1 Depletion on the Self-Renewal of Normal HSC VersusLeukemic Cells

Terminal differentiation is critically dependent on lineage-determiningtranscription factors such as PU.1. PU.1, like other lineage-determiningDNA binding factors, demonstrates both transcription repression andtranscription-activating functions, determined by interactions witheither corepressors versus coactivators. The murine PUER cell-line isderived from Pu.1 knock-out cells, which have been transduced with aretroviral vector which expresses Pu.1 fused to the estrogen-receptor.Addition of the estrogen agonist tamoxifen (OHT) to these cells causesPu.1 to be functionally reintroduced into the cell through translocationinto the nucleus, and triggers terminal differentiation.

FIG. 6) Pu.1 induced terminal differentiation involves orderly andsequential repression of genes associated with self-renewal (HoxB4,Bmi-1, c-Kit), followed by activation of genes associated with terminaldifferentiation (Mcsfr, Gmcsfr, F4/80) (latter data not shown).

Example 6 The Phenotypic Consequences of DNMT1 Depletion Depend on theDifferentiation Chronology of the Cell

FIG. 7A) The effect of decitabine on proliferation of PUER is dependenton the timing of decitabine addition in relationship to Pu.1 activation(Pu.1 is functionally activated by adding OHT to the cells). Addingdecitabine concurrent with Pu.1 activation impaired Pu.1-mediatedterminal differentiation and preserved some cell proliferation. However,adding decitabine 6 h after Pu.1 activation did not. FIG. 7B) Concurrentdecitabine and Pu.1 activation inhibited differentiation, but decitabine6 hrs after Pu.1 activation did not. Flow cytometry was used to measureF4/80 as a marker of terminal macrophage differentiation and c-Kit asmarker of stem-cells. Cell morphology was consistent with theflow-cytometry data (data not shown). FIG. 7C) Decitabine additionconcurrent with Pu.1 activation prevented the first step in terminaldifferentiation of pro-self-renewal gene repression. However, decitabineaddition after pro-self-renewal gene repression had occurred (6 hoursafter OHT) increased pro-differentiation gene expression.

Therefore, the phenotypic consequences of DNMT1 depletion criticallydepend on the differentiation chronology of the cell. By preventing thefirst step in differentiation, which is repression of pro-self-renewalgenes, decitabine can increase self-renewal even in adifferentiation-promoting context. These findings are a reasonableexplanation for our published observation that decitabine increasesself-renewal of normal HSC (Milhem, et al. 2004 Blood 103(11):4102-10).In contrast, increased differentiation in response to decitabine that isadded after self-renewal genes have already been repressed, resemblesthe effect of decitabine on leukemia cells. The opposite effects ofdecitabine on HSC versus leukemia cells could be proposed to resultbecause leukemia is arrested differentiation in progress, distinct fromself-renewal of HSC.

Example 7 Molecular Events When a Leukemia First-Hit Event (Disruptionof Runx1) Inhibits Pu.1 Mediated-Differentiation

Runx1 disruption, by congenital or acquired Runx1 mutations andchromosome translocations, is one of the most frequent genetic events inmyelodysplasia and leukemia.

Knock-down of Runx1 expression in PUER cells did not preventPu.1-mediated repression of pro-self-renewal genes, but did inhibitPu.1-mediated activation of pro-differentiation genes. Using lenti-viralshRNA delivery, Runx1 expression was knocked down in PUER cells, anddecreased Runx1 expression was confirmed by Western blot (between 25-50%of control levels), in different PUERshRunx1 clones (data not shown).PUERshRunx1 cells did not terminally differentiate in response to Pu.1activation (+OHT) (data not shown). FIG. 8) In PUERshRunx1 cells, thefirst-step in Pu.1 mediated differentiation of pro-self renewal (Bmi-1,HoxB4, c-Kit) repression is intact. However, Runx1 knock-down preventedthe next chronological step in differentiation—activation ofpro-differentiation genes (F4/80, mcsfr, gmcsfr).

Example 8 Leukemia Cells From Patients Conform to the Model

To examine the clinical relevance of the above findings regarding arrestin differentiation-transit, the levels of lineage-specifying factorswere measured in primary leukemia cells from patients. Keylineage-specifying transcription factors in hematopoiesis include PU.1(required for macrophage and B-lymphocyte production), CEBPα and CEBPεrequired for neutrophil production and GATA1 for erythroid production(Iwasaki, H., et al. 2006 Genes Dev 20:3010-3021). The levels of thesefactors increase during differentiation into the respective lineages.The expression levels of these factors were measured in bone marrowaspirate cells from normal donors, patients with low-riskmyelodysplastic syndrome (MDS—a clonal hematologic disorder which oftenprecedes AML) and patients with high-risk myelodysplastic syndrome andacute myeloid leukemia (high-risk disease). The low-risk MDS patientbone marrow aspirates contain abnormally differentiated and increasedimmature cells, but <5% myeloblasts. In the high-risk patient bonemarrow aspirates, the average percentage of myeloblasts was 40%. Thehigh-risk samples, although morphologically the least mature, had thehighest levels of the myeloid lineage-specifying factors CEBPα, CEBPεand PU.1 (FIG. 9A). GATA1 levels were not significantly increased in thedisease samples compared to normal controls. The clinical annotation ofthe samples analyzed is shown (FIG. 9B).

Micro-array gene-expression data from 54 AML patients demonstrated asimilar pattern of increased expression of lineage-specifying factors inAML samples compared to normal bone marrow cells (Yagi, T., et al. 2003Blood 102:1849-1856).

Unlike DNA mutation or chromosome aberration, it is not immediatelyobvious that a difference in promoter CpG methylation between amalignant and a normal sample is abnormal, since promoter CpGmethylation varies among different tissues and with stage ofdifferentiation. Therefore, classifying promoter CpG sites by themethylation changes that occur during normal differentiation couldimprove interpretation of DNA methylation studies in cancer andleukemia. In particular, such classification could illuminate the roleof differentiation in malignant cell-specific patterns of promoter CpGmethylation.

To address this issue, a promoter CpG methylation micro-array was usedto classify CpG sites by hypo-methylation, hyper-methylation, or nosignificant methylation change during normal myeloid differentiation.This classification was applied to a study of promoter CpG methylationpatterns in primary myelodysplastic syndrome (MDS) and acute myeloidleukemia (AML) cells. In primary MDS cells, methylation atdifferentiation responsive CpGs was the inverse of that in normal CD34+hematopoietic stem and progenitor cells (HSPC), with hypo-methylation ofCpG that are normally hyper-methylated in CD34+ HSPC and vice-versa. Inhigh risk (≧5% myeloblasts) MDS/AML samples, and AML cell linesincluding CD34+ AML cell lines, this pattern was further exaggerated.This difference between normal HSPC and MDS/AML cell epigenetics couldcontribute to contrasting differentiation fates in response to drugsthat inhibit chromatin-modifying enzymes (FIG. 9C).

This observation provides insight into leukemogenesis and MDS/AMLbiology. Promoter CpG methylation reflects differentiation stage orcontext. The pattern of promoter CpG methylation in the MDS/AML cells isconsistent with a differentiation context that is more advanced orcommitted than normal HSPC. This possibility is clarified by the highexpression of the key lineage-specifying transcription factors PU.1 andCEBPα in MDS/AML cells compared to normal hematopoietic stem andprogenitor cells. Therefore, the pattern of promoter CpG methylationsuggests, and is likely one dimension of, a lineage-committeddifferentiation context of MDS/AML cells.

FIG. 9. Leukemia cells from patients are differentiation-impaired afterlineage-commitment. A) High risk MDS and AML cells express high levelsof lineage-specifying factors (measured by RQ-PCR). Samples are bonemarrow from normals, patients with low risk MDS (<5% myeloblasts), andhigh risk MDS/AML (>5% myeloblasts). B) WHO classification and detectedchromosome abnormalities in the analyzed samples. C) In MDS and AML bonemarrow, the direction of methylation change at differentiationresponsive CpG sites mimics that seen during normal differentiation,only exaggerated. Methylation levels are represented by a β-valuebetween 0 (unmethylated) and 1 (fully methylated). Promoter CpG siteswere classified into 3 categories: CpG sites that undergo a significant(p<0.001, t-test) increase in methylation from normal stem andprogenitor cells (HSPC, NCD34+) to normal mature cells (NBM) (108 CpG,‘hypomet. in NCD34+’) (left box plots); CpG sites that undergo asignificant (p<0.001, t-test) decrease in methylation from NCD34+ to NBM(162 CpG, ‘hypermet. in NCD34+’) (middle box plots); CpG sites that donot undergo a change in methylation between NCD34+ and NBM (1236 CpG,‘no met. change during n. diffn.’) (right box plots). Asterixesrepresent statistically significant differences between the median inthe sample group compared to the NBM group (Kruskal-Wallis). Actualp-values are: ‘Hypo-met in NCD34’-NBM v NCD34<0.0001, NBM vLoRisk<0.0001, NBM v HiRisk<0.0001. ‘Hyper-met in NCD34’-NBM v NCD34<0.0001, NBM v LoRisk=0.024, NBM v HiRisk<0.0001. ‘No Change’-NBM vNCD34 <0.0001^(#), NBM v LoRisk NBM v LoRisk=0.024, NBM v HiRisk<0.0001,NBM v HiRisk<0.0001. NCD34=CD34+ cells isolated from normal bone marrow(n=9), NBM=normal whole bone marrow (n=42), LoRisk=bone marrow fromlow-risk MDS patients (n=27), HiRisk=bone marrow from high-risk MDS/AMLpatients (n=130). Box-plot boundaries=inter-quartile range, horizontalline=median, ‘+’ = mean, whiskers=range of values, small boxes=out-lyingvalues. ^(#)CpG were classified as ‘no change in met’ based on a t-testto compare means between NCD34 and NBM, whereas a Kruskal-Wallis test tocompare medians is used here.

Example 9 A Model of Normal Differentiation and Leukemia Self-RenewalThat Explains Why DNMT1 Depletion Has Opposite Effects on Normal andMalignant Cells

Differentiation mediated by a lineage-specifying transcription factor orby cytokines (Milhem, M., et al. 2004 Blood 103:4102-4110) requiresorderly repression of stem-cell associated genes followed byupregulation of differentiation-fate associated genes. The repression ofstem-cell associated genes requires chromatin modifying proteins such asDNMT1. Therefore, DNMT1 depletion, by preventing this initial phase,prevents differentiation and maintains self-renewal of dividing normalstem-cells, even in differentiation inducing conditions (Milhem, M., etal. 2004 Blood 103:4102-4110) (data not shown).

In a substantial number of AML cases, differentiation arrest occursafter lineage-commitment (hence AML cells express lineage markers andhigh levels of lineage-specifying factors) and after the phase ofstem-cell associated gene repression. Therefore, the self-renewal(proliferation at the same level of differentiation) of the leukemiacells is an aberrant persistence of the cell-division of differentiatingcells, which is usually terminated by completion of the differentiationprocess. The differentiation impairment which maintains this abnormalself-renewal, although it may be initiated by genetic abnormalities, isfinally mediated by epigenetic mechanisms that aberrantly repress genesnecessary for differentiation. Therefore, DNMT1 depletion to antagonizethe transcription repression machinery restores the differentiation forwhich these cells are poised and thereby terminates the abnormalself-renewal. Observations in cancer cell lines representing a spectrumof cancer histologies indicate that the proposed model is relevant inmany cancers.

Example 10 Translation of These In Vitro Observations Into Effective InVivo Therapy Must Overcome a Number of Pharmacologic Obstacles

The MA9 xenograft model of human leukemia was treated withintra-peritoneal decitabine alone (1mg/m² 3×/week). Decitabine increasedsurvival by approximately 20%, but all mice succumbed to leukemia (FIG.10). This poor result, which did not reflect the in vitro findings,highlights the pharmacologic barriers that limit the in vivo activity ofdecitabine and hinder successful translation of very promising in vitrofindings. CDA is the most important pharmacologic barrier to successfultranslation of in vitro findings into in vivo therapy. The inventionaddresses this barrier to effective clinical translation of the in vitroobservations.

An antagonist of the decitabine degrading enzyme cytidine deaminase(CDA)—tetrahydrouridine (THU). THU is a pyrimidine analogue thatinhibits CDA (Ki 3-5×10-8M). THU has a benign toxicity profile,well-characterized PK, and has been administered to humans byintravenous (IV), sub-cutaneous (SQ), and oral (PO) routes in a numberof clinical trials (Ho, D. H., et al. 1978 J Clin Pharmacol 18:259-265;Kreis, W., et al. 1977 Cancer Treat Rep 61:1347-1353; Kirch, H. C., etal. 1998 Exp Hematol 26:421-425; Yue, L., et al. 2003 Pharmacogenetics13:29-38; Gilbert, J. A., et al. 2006 Clin Cancer Res 12:1794-1803;Bhojwani, D., et al. 2006 Blood 108:711-717; Liu, Z., et al. 2007Nucleic Acids Res 35:e31).

Example 11 Pharmacologic Obstacle: Inter-Individual Variation inResponse to Cytosine Analogues

The AA genotype of CDA is associated with poor outcome in response tonucleoside analogue therapy. In humans, CDA is subject to anon-synonymous single nucleotide polymorphisms (SNP) (RS2072671) whichproduces an A→C transition in the ancestral allele, changes lysine toglutamine at amino-acid position 27 and decreases CDA activity 3-fold(Gilbert, J. A., et al. 2006 Clin Cancer Res 12, 1794-1803; Kirch, H. C.et al. 1998 Exp Hematol 26, 421-425; Yue, L., et al. 2003Pharmacogenetics 13, 29-38). In the human hapmap (publicly availablehaplotype map of the human genome), 40% of Caucasians are homozygouswith the AA genotype, 50% heterozygous with AC genotype and 10%homozygous with CC genotype (AA frequency is>90% in Asians andAfricans). Since CDA can mediate cancer resistance to cytosineanalogues, it was examined whether these different genotypes of CDApredicted clinical outcomes in MDS and AML patients treated withcytosine arabinoside (ARA-C), 5-azacytidine, or decitabine.

The AA genotype of CDA, which has greater enzymatic activity, isassociated with early relapse during cytosine analogue therapy but notwith non-cytosine analogue therapy. It was hypothesized that the moreenzymatically active AA genotype of CDA, by further limiting theactivity of cytosine analogues (ARA-C, 5-azacytidine or decitabine),could cause primary resistance or early relapse (relapse within 2 years)in MDS or AML patients treated with these drugs, but not in MDS or AMLpatients not being treated with cytosine analogues. Using the HumanNS-12BeadChip (Illumina, San Diego, Calif.) and allele-specific DNAsequencing, 24 month survival was stratified by CDA genotype in 81 MDSand AML patients treated at the Cleveland Clinic. As expected, the AAgenotype was associated with decreased 24 month survival (p=0.05 Cox) inpatients treated with cytosine analogues (data not shown). Numbers forthe CC genotype are small, but it could be proposed that CC genotype mayincrease mortality from toxicity, but decrease relapse mortality. Inpatients who were not treated with cytosine analogues (these patientsreceived arsenic or gemtuzumab), CDA genotype did not influence survivaloutcome (data not shown).

This data may explain the poorer leukemia outcomes seen in non-Caucasianpopulations (who are>90% AA genotype), and indicates that thecombination decitabine-tetrahydrouridine (THU) agent contemplated hereinshould be a significant advance in MDS and AML therapy (and possiblytherapy for other cancers) in all populations, since by inhibiting CDA,it is likely to address a major mechanism that underlies poor outcomewith cytosine analogues.

Early relapse of leukemia is believed to represent selection forchemo-resistant clones, whereas late relapse may represent the emergenceand evolution of leukemia stem-cells that were quiescent and unexposedto induction and consolidation chemotherapy. The combination ofdecitabine with THU has the potential to address both forms of relapse.Early relapse can be reduced by improving the pharmacologic profile(increasing time above threshold concentration, decreasinginter-individual variation) of decitabine. The lack of toxicity of theproposed approach, which should facilitate chronic, long-term therapy,may address late relapse, since even relatively quiescent LSC should atsome point be exposed to chronically administered decitabine.

Example 12 Pharmacologic Obstacle: The Very Brief In Vivo Half-Life ofDecitabine

As seen above, CDA genotype is associated with significantly differentoutcomes in response to cytosine analogue therapy. This reflects thevery large influence CDA has on cytosine analogue therapy in vivo: (i)Because of destruction by the enzyme CDA, the in vivo half-life of IVdecitabine is <20 minutes, compared to an in vitro half-life of 5-9hours (Liu, Z., et al. 2006 Rapid Common Mass Spectrum 20:1117-1126). Inorder to exploit decitabine's unique quality amongst nucleosideanalogues, its ability to deplete DNMT1 at low concentrations, thepharmacologic objective of therapy is to maximize time-above-thresholdconcentration for depleting DNMT1 (>0.2 μM), while avoiding the highpeak levels (>1 μM) that damage DNA (the DNA damage that occurs athigher levels causes toxicity that limits the cumulative dose of drugand is, therefore, counter-productive). To examine how different routesof administration might serve this pharmacologic objective, decitabinepharmacokinetics were studied in the non-human primate baboon model.

FIG. 11: Subcutaneous (SQ) and oral decitabine produces lower peaklevels but a longer time-above-threshold for DNMT1 depletion (˜0.2 μM)than IV decitabine. Baboons were treated with IV decitabine 0.5 mg/kg (2animals), SQ decitabine 0.5 mg/kg (2 animals), or oral decitabine 10mg/kg (2 animals). Blood was collected for pharmacokinetic (PK) analysisat 7 time-points per animal and decitabine levels measured by LC-MS(Liu, Z., et al. 2006 Rapid Common Mass Spectrum 20:1117-1126). Thisdata demonstrates: (i) SQ and oral administration can produce a longertime-above threshold concentration (0.2 μM) for depleting DNMT1 than IVdecitabine. Indeed, intravenous (IV) decitabine has an abbreviatedhalf-life of <20 mins; (ii) IV administration, but not SQ or oral, canproduce peak levels (>1 μM) associated with DNA damage; (iii)Significant inter-individual variability in PK that is most prominentwith oral administration of the drug.

Example 13 Pharmacologic Obstacle: Malignant Cell Resistance toDecitabine is Pharmacological Rather Than Biological

Resistance to the effects of decitabine appears to be pharmacologicrather than biological, i.e., resistance is associated with a failure ofdecitabine to deplete DNMT1, rather than continued proliferation despitedepletion of DNMT1.

FIGS. 12A and B) Using the Q-Dot immunofluorescence assay for DNMT1levels, decitabine-sensitive cell lines demonstrate DNMT1 depletion fromthe nucleus (in comparison with decitabine-resistant cell lines).However, the decitabine-resistant cell-line PC3 demonstrated persistentDNMT1 expression. This indicates that the mechanism of resistanceinvolves decreased decitabine entry into the cell, increased decitabinedestruction CDA, or decreased activation by DCK.

Example 14 Non-Clinical Pharmacokinetics of Decitabine Alone and inCombination With THU

Pharmacokinetics of decitabine in non-human primates, poor oralbio-availability of decitabine in non-human primates: thepharmacokinetics of decitabine were assessed in two baboons followingoral, SQ and intravenous administration. This experiment demonstratedthat the oral bioavailability of decitabine in these two baboons rangedfrom approximately 0.2% to 7.0% (FIG. 14). Decitabine was administeredto two baboons IV (0.5 mg/kg), SQ (0.5 mg/kg) and orally (10 mg/kg).Blood was collected at 7 time-points and plasma concentrations weredetermined using an LC/MS/MS method adapted from a previously publishedmethod 26. This data demonstrates: (i) that SQ and oral administrationcan produce a longer time-above-threshold concentration (˜0.2 μM) fordepleting DNMT1 than IV decitabine; (ii) that at the doses studied, IVadministration, but not SQ or oral, can produce peak levels (>1 μM)associated with DNA damage; and (iii) significant inter-individualvariability in decitabine exposure that is most prominent with oraladministration of the drug.

Example 15 Substantial Inter-Individual Variation in oralBio-Availability of Decitabine in Baboons

Inter-individual variability was likewise seen in seven baboons thatreceived 10 mg/kg decitabine orally, with the PK being measured at 6 or7 time-points (FIG. 15).

Example 16 Administation of Oral THU Prior to Oral Decitabine Addressesthe Substantial Inter-Individual Variability in Oral Bio-Availability ofDecitabine

High and low responder baboons (PA7470 and PA7484, respectively) weregiven 10 mg/kg decitabine alone, 0.5 mg/kg decitabine and 20 mg/kgtetrahydrouridine (THU) concurrently, or 0.5, 2, or 5 mg/kg decitabine60 minutes after 20 mg/kg tetrahydrouridine (THU). Cmax (ng/mL), Tmax(min), and AUClast (min*ng/mL) were measured and are shown in Table 1,below, and FIG. 16. Following the oral administration of drugs, theplasma concentration generally reaches, in principle, a single,well-defined peak (Cmax) at the time of Tmax. AUClast refers to the areaunder a plotted plasma concentration-time curve (not shown) at the lastrecorded timepoint.

TABLE 1 Cmax Tmax AUClast Animal Decitabine (mg/kg) (ng/mL) (min)(min*ng/mL) PA7470 10 mg/kg 51.59 60 6278.775 PA7470 0.5 mg/kg + 20mg/kg 4.24 30 347.25 THU (0 min) PA7470 0.5 mg/kg + 20 mg/kg 3.06 66356.81 THU (60 min) PA7470 5 mg/kg + 20 mg/kg 48.54 189 7534.665 THU (60min) PA7484 10 mg/kg 2.33 45 190.35 PA7484 2 mg/kg + 20 mg/kg 34.01 1801729.15 THU (60 min) PA7484 5 mg/kg + 20 mg/kg 65.87 120 5621.05 THU (60min)

Of note, the administration of THU and decitabine increases the oralbio-availability of decitabine in the poor responder baboon andconverges the PK between poor and good responder baboons (4^(th) and7^(th) values in final column) in comparison with decitabine alone(1^(St) and 5^(th) values in final column). In effect, combining THUwith oral decitabine increased the exposure of decitabine approximately60-fold in an animal with relatively low bioavailability andapproximately 2-fold in an animal with relatively high bioavailability,such that the extensive inter-individual variability in decitabineexposure was substantially reduced.

Example 17 Identifying the Dose of THU to Use to Increase OralBio-Availability

Baboons received varying doses (2 mg/kg or 20 mg/kg) oftetrahydrouridine 60 minutes before a dose (5 mg/kg) of decitabine. Twobaboons (PA7484 and PA7470) were administered oral THU 20 mg/kg (500mg/m²) 60 minutes prior to oral decitabine 5 mg/kg, or decitabine alone.After a wash-out period of greater than 2 weeks, these same two animalswere administered oral THU 2 mg/kg (50 mg/m²) 60 minutes prior to oraldecitabine 5 mg/kg, or oral decitabine alone. Unlike 20 mg/kg (500mg/m²), 2 mg/kg (50 mg/m²) of THU was insufficient to achieve the targetplasma concentration of decitabine of >50 ng/mL (FIG. 17). Both doses oftetrahydrouridine significantly increased decitabine oralbio-availability, but this increase was significantly greater with thehigher dose (500 mg/m², or, 20 mg/kg) of tetrahydrouridine.

Example 18 Identifying the Optimal Timing Between Oral THU and OralDecitabine Administration (Oral Bio-Availability of Decitabine UponConcurrent vs. Prior Administration of Tetrahydrouridine)

High-responder and low-responder baboons each received either 20 mg/kg(500 mg/m²) tetrahydrouridine (THU) orally 60 minutes before oraladministration of 5 mg/kg decitabine or concurrently with thedecitabine. Decitabine levels were measured at various timepoints byLC-MS.

Notably, when the tetrahydrouridine was given orally concurrently withthe decitabine, the oral bio-availability of the latter exhibited asignificant drop-off in comparison to administration of thetetrahydrouridine 60 minutes prior to decitabine (data not shown).

Example 19 Confirmation That Oral THU Increases the OralBio-Availability of Decitabine Approximately 4-Fold, and Decreases theSubstantial Inter-Individual Variation in Decitabine OralBio-Availability

In seven baboons, the administration of THU prior to decitabineincreased the oral bioavailability of decitabine approximately 4-foldand decreased inter-individual variation in decitabine pharmacokinetics.Seven baboons were treated with decitabine alone 10 mg/kg by oralgavage, or after a wash-out period of at least 2 weeks, with THU 20mg/kg by oral gavage followed by decitabine 5 mg/kg by oral gavage. In 7animals administered oral decitabine alone at 10 mg/kg, the mean AUC was1604 and the median AUC was 463 min*ng/ml (FIG. 18A). In these same 7animals administered THU 20 mg/kg followed by half the dose ofdecitabine (5 mg/kg), the mean AUC was 2820.914 and the median AUC was2284 min*ng/ml (FIG. 18A). Therefore, administering oral THU prior tooral decitabine produced a fold-increase in oral bioavailability ofapproximately 10-fold when considering medians, and approximately3.5-fold when considering means. The largest increases in AUC withco-administration were seen in the animals with low AUCs with decitabinealone (FIG. 18B). Therefore, the large inter-individual variation seenwith decitabine alone, represented by the separation between mean andmedian AUC, was substantially dampened by co-administration of THU withdecitabine (FIG. 18A).

Mice received varying doses (30 mg/kg, 15 mg/kg, and 7.5 mg/kg) oftetrahydrouridine 30 minutes before a dose (16 mg/kg) of decitabine.Decitabine levels were measured by LC-MS. 50 mg/m² tetrahydrouridineadministered prior to the administration of decitabine resulted in anapproximately 5-fold (certainly pharmacologically significant) increasein decitabine oral bio-availability (data not shown). A slight drop-offwas observed in decitabine oral bio-availability measured when thedosage of THU was decreased from 50 to 25 mg/m².

Of note, a similar cytidine deaminase expression pattern is found inhumans and mice (data not shown). This gene expression data can beobtained from the public gene expression database GenAtlas.

Example 20 Phase 1/2 Study of Chronic Low Dose IV Administration ofDecitabine in SCD

Based on previously conducted trials, in which no clinically significantadverse events occurred, a chronic administration study was conducted toidentify the toxicity and effectiveness of repeated decitabine dosingover 36 weeks (9 months) in 7 subjects with HU refractory SCD (DeSimone,J., et al. 2002 Blood 99:3905-3908). Decitabine was administered by I.V.infusion at 0.3 mg/kg/day, 5 consecutive days per week for 2 weeks,followed by a 4-week observation period. If the absolute neutrophilcount (ANC) dropped below 1×109/L, the dose was reduced by 0.05 g/kg/dayin the next 6- week cycle. An optimal drug dose was obtained for eachsubject, and resulted in an elevated HbF without neutropenia (ANC nadir>1.5×109/L).

Pharmacodynamic effects: the average HbF and average maximal HbF levelsattained during the last twenty weeks of treatment for the 7 SCDsubjects were 13.93±2.35% and 18.35%±4.46%, respectively (from abase-line of 3.12%±2.75%). The average and average maximal hemoglobinvalues were 8.81±0.42 g/dL and 9.7±0.53 g/dL, respectively (from abase-line of 7.23±2.35 g/dL) (Table 2, below).

TABLE 2 Hemoglobin and HbF levels before and during the last 20 of 36wks of treatment with decitabine HbF (%) Total Hemoglobin (g/dL) SubjectPre Avg Max Pre Avg Max 1 0.8 12.40 ± 1.25 14.4 6.2 9.05 ± 0.48 9.6 26.8 14.55 ± 1.32 16 3 8.2 9.37 ± 0.60 10.3  3 1.4 12.75 ± 2.28 17.2 6.08.34 ± 0.55 9.5 4 0.6 10.80 ± 2.05 14.4 7.2 8.28 ± 0.52 9.0 5 2.9 16.42± 2.81 24.6 8.0 8.91 ± 0.57 9.6 7 6.2 16.70 ± 2.55 23.2 7.8 8.92 ± 0.7910.4  Mean ± SD 3.12 ± 2.75 13.93 ± 2.35 18.35 ± 4.46 7.32 ± 0.94 8.81 ±0.42 9.73 ± 0.53

Individual maximal F-cell numbers during the trial ranged from 58-87%(i.e., an average over all 7 subjects of 69±10.12%).

Hematologic side-effects and toxicity: despite periodic depressions inANCs, which occurred 5 to 6 weeks after beginning each treatment cycle,the average ANC during the last 20 weeks of treatment (4.2±1.35×109/L)was not significantly different from the pretreatment average(4.6±1.56×109/L). The ANCs of two HU non-responder subjects never fellbelow 2.0×109/L and the nadirs, which occurred at 5-6 weeks of eachcycle, generally remained above 3.0×109/L. No clinical sequelae of bloodcount changes occurred.

Non-hematologic toxicity: no non-hematologic toxicity occurred. Patientsdid not require anti-emetics.

Example 21 Phase 1/2 Study of SQ Administration of Decitabine

A Phase 1/2 study was initiated using decitabine given by the SQ routein order to assess the safety of decitabine given by the SQ route, toproduce cumulative increases in fetal and total hemoglobin throughweekly administration, and to explore the mechanism by which decitabineincreases HbF (Saunthararajah, Y., et al. 2003 Blood 102:3865-3870).Eight subjects with multiple clinically significant complications of SCDwere treated. Decitabine was administered at 0.2 mg/kg SQ 1 to 3 timesper week in 2 cycles of 6-week duration with a 2-week interval betweencycles. In cycle 1, drug was administered twice per week on 2consecutive days. If the patient achieved an F-cell percentage (%F-cells) of at least 80% during cycle 1, the dose frequency was reducedto once per week in cycle 2. If the highest % F-cells during the cycle 1was less than 80%, the dose frequency was increased to 3 times per weekin cycle 2.

Pharmacodynamic effects: all subjects demonstrated statisticallysignificant increases in HbF expression (FIG. 13). DNA methylationanalysis of the γ-globin promoter in DNA isolated from bone marrowaspirate cells was demonstrated post-treatment hypomethylation at thislocus (data not shown). At higher doses, decitabine is known to becytotoxic. To determine if this dose and schedule of decitabine wascytotoxic, bone marrow morphology was evaluated by independent andblinded hematopathology review of pre-treatment and post-treatment bonemarrow aspirates. There was no decrease in bone marrow cellularity. Anincrease in erythroid cells and megakaryocytes was noted (data notshown). Flow cytometric analysis of propidium iodine stained freshmarrow aspirate cells did not demonstrate an increase in the sub-G1apoptotic fraction (data not shown).

Hematologic side-effects and toxicity: one patient had (National CancerInstitute/Cancer Therapy Evaluation Program (NCI/CTEP) grade 4neutropenia (nadir ANC 0.4×103/μl ), two had grade 3 neutropenia (nadirANC 0.8×103/μl). Neutropenia recovered within 7 days of the lastdecitabine dose. Neutropenic fever did not occur. Platelet countsincreased in all subjects during treatment. The highest platelet countwas 877×109/L. No clinical sequelae to these blood count changesoccurred.

There was an inverse relationship between platelet and neutrophil counts(data not shown). The changes in bone marrow morphology and in vitrostudies with non-cytotoxic levels of decitabine indicate that themechanism of decreased neutrophils and concurrent increased platelets isaltered hematopoietic stem cell differentiation (Saunthararajah, Y., etal. 2003 Blood 102:3865-3870).

Four patients consented to serial bone marrow aspirate and biopsyanalysis before and each 6-wk cycle of decitabine treatment.

TABLE 3 Results of bone marrow aspirate and biopsy analysis.Pre-Treatment After Cycle 1 After Cycle 2 M:E MK/ M:E MK/ M:E MK/ ID#Cellu ratio LPF Cellu ratio LPF Cellu ratio LPF UPN1 Hyper 0.8 4 Hyper0.7 10 Hyper 0.6  13 UPN3 Hyper 0.8 2-3 Hyper 0.3   6 UPN5 Norm 2   2Norm 0.8  6 Norm 0.5   6 UPN7 Hyper 1   1 Hyper 0.3  8 Hyper 0.03 13

There was no decrease in marrow cellularity upon hematopathology review(Table 3, above), which was blinded to treatment status. In UPN3, theinterim bone marrow aspirate was technically unsuccessful.

Non-hematologic toxicity: NCI Toxicity Criteria were used to assesstoxicity. No local toxicity occurred at SQ injection sites. Nonon-hematologic toxicity occurred. No subjects described nausea,vomiting, diarrhea, constipation, or decreased appetite.

Efficacy: total Hb increased from 7.6±2 to 9.6±1.8 (mean±2 SD ofpre-treatment to peak Hb, paired t-test p<0.001). Both the absolutereticulocyte count (ARC) (p=0.0006) and total bilirubin (p=0.01,2-tailed paired t-test) decreased during treatment. The ARC correlatedinversely with tHb (p<0.0001). In SCD, abnormal exposure of moleculessuch as phoshphatidyl-serine on the RBC surface and adhesion of RBC toendothelial cells/endothelial damage can trigger coagulation andinflammatory pathways. RBC adhesion to both TSP and laminin decreasedfollowing cycle 1 (p<0.005). In agreement with previous reports (Tomer,A., et al. 2001 J Lab Clin Med 398-407; Francis, R. B., Jr. 1989Haemostasis 19:105-111; Peters, M., et al. 1994 Thromb Haemost71:169-172), increased levels in markers of active coagulation,Thrombin-antithrombin (TAT), F1+2 and D-dimers, were noted at baseline.Treatment decreased D-dimer levels, a measure of fibrinolysis ofcross-linked fibrin (p<0.04), while markers of thrombin generation, TATand F1+2, decreased, but not to a statistically significant extent. Theadhesion molecule soluble VCAM (sVCAM-1) and von Willebrand factorpeptide (VWFpp) are released from damaged endothelial cells, levels ofboth molecules decreased with treatment (p<0.05). C-reactive protein(CRP), a marker of inflammation, was elevated at baseline and, althoughthere was a downward trend with therapy, it was not statisticallysignificant (p=0.18) (see Table 4, below).

TABLE 4 Changes in surrogate clinical endpoints. Post Post NormalPretherapy Cycle 1 P* Cycle 2 P** Range Measures of Adhesion to 1570 ±170  690 ± 150 <0.001 910 ± 160 <0.001 <60 RBC adhesion TSP to(RBCs/mm²) endothelium Adh. to 3470 ± 500  1950 ± 300  0.004 1570 ± 210 <0.001 <250 laminin (RBCs/mm²) Measures of D-Dimer 490 ± 90  320 ± 50 0.02 300 ± 50  0.03 <400 thrombin (ng/mL) generation TAT (ug/L) 7.0 ±1.7 8.6 ± 2.3 0.15 5.2 ± 0.9 0.11 1.0-4.1 and F1 + 2 (nmol/l) 1.75 ±0.22 1.56 ± 0.16 0.23 1.41 ± 0.15 0.051 0.04-1.1  fibrinolysis Measureof CRP (mg/dL) 1.25 ± 0.27 1.19 ± 0.34 0.80 0.82 ± 0.26 0.18 <0.7inflammation Measures of sVCAM (ng/ml) 1170 ± 140  930 ± 100 0.01 840 ±100 0.02 395-714 endothelial VWFpp (u/dL) 196 ± 26  156 ± 28  0.015 144± 13  0.049  74-153 cell damage Values are mean ± SE; paired 2-tailedt-test. P* = significance of change from pre-therapy to post-cycle 1.P** = change from pre-therapy to post-cycle 2.

Example 22 Chronic Decitabine Administration to Seriously Ill SCDPatients (Saunthararajah, Y., et al. 2008 Br J Haematol 141:126-129)

Previous studies of decitabine as a potential disease-modifying agentfor sickle cell disease (SCD) were not designed to demonstrate clinicaleffectiveness. In four SCD patients with severe acute illness on abackground of chronic clinical deterioration over the preceding years ormonths, decitabine (0.1-0.2 mg/kg 1-2×/week) was administered off-labelfor periods beyond 12 months. The off-label use of decitabine in SCD wasto provide direct benefit to these patients and not for research.Decitabine was considered because of clinical deterioration andlife-threatening complications despite HU therapy, erythropoietin forrelative reticulocytopenia (hemoglobin <9 g/dl & reticulocytes≦250×109/L), decreased availability, and increased transfusion risksfrom ≧5 red blood cell (RBC) allo-antibodies and auto-antibodies, andineligibility for available protocol therapy.

Hemoglobin increases of >1.5 g/dl occurred within 2-4 weeks with maximumhemoglobin increases of 3.5-5g/dl. Hemoglobin increased through anincrease in reticulocytes and an increase in fetal hemoglobin (theincrease in hemoglobin was not explained by increase in fetal hemoglobinalone). Generally, reticulocyte counts increased during the first 2-8weeks of therapy. Reticulocyte trends reversed after hemoglobinlevels >9g/dl. This was most obvious in Patient A, who was not receivingexogenous erythropoietin. Durable symptom and performance statusimprovement during 4-12 months of follow-up contrasted with severe anddeteriorating pre-decitabine trends.

Of note, all patients had severe acute illness on a background ofchronic deterioration and progressive anemia over the preceding years ormonths; the follow-up period ranging from 4-12 months alloweddocumentation of durable clinical improvement that contrasted obviouslywith clinical status and trends in the preceding months; although 3 ofthe 4 patients were on concurrent erythropoietin, it had beenadministered at stable doses for more than 6 months with progressiveanemia and recurrent severe anemia exacerbations; although 2 of the 4patients received transfusions during decitabine therapy, these do notexplain the durable increases in hemoglobin and eventual transfusionindependence. The severe and complicated clinical circumstance in thesepatients is not typically represented in clinical trials. Therefore,this description can complement the clinical studies and provideadditional guidance regarding dose, schedule, anticipated toxicities andinclusion criteria.

Example 23 Pharmacodynamics of Decitabine Alone and in Combination WithTHU

Significant inter-individual variability in pharmacodynamic responses inbaboons. The biologic relevance of differences in pharmacokineticresponses to oral decitabine were examined in a different set ofbaboons, in which large differences in individual pharmacodynamicresponses (fetal hemoglobin expression) and biologic activity(neutrophil counts) were noted (Table 5, below).

TABLE 5 Pharmacodynamic responses following SQ and oral administrationof decitabine to baboons.

Fetal hemoglobin expression, HbF %, pre and post treatment with theindicated dose of decitabine. Decreases in neutrophil counts, anotherexpected biological effect of decitabine, paralleled the increases inHbF %. Oral treatment was with decitabine or a slightly modifieddecitabine (decitabine-mesylate—Dac-m). Inter-individual variability inpharmacodynamic responses to the same dose of oral decitabine arehighlighted by the boxes.

Example 24 The Administration of THU Prior to Decitabine DecreasesInter-Individual Variability in Decitabine

Similarly, administration of oral THU prior to oral decitabine in twoother baboons (PA 6974 and 7001) produced a substantial increase in thepharmacodynamic responses (fetal hemoglobin expression) to oraldecitabine (Table 6, below).

TABLE 6 Pharmacodynamic responses (fetal hemoglobin expression, HbF %)were substantially enhanced by administering oral decitabine after oralTHU. Dose THU oral Pre- Post- Animal Drug Route mg/kg/d 20 mg/kg HbF %HbF % 6974 Dac Oral 9.35 − 10.3 40.5 Dac Oral 0.3  +  3.2 28.4 7001 DacOral 9.35 −  6.6 17.4 Dac Oral 1.42 +  6.2 61.6

Decitabine was administered at the doses indicated in two consecutiveblocks of 5 days each. THU at the indicated doses was administered 60minutes before each decitabine dose.

1. A composition for oral administration comprising about 10 to about150 mg/m² decitabine and about 100 to about 500 mg/m² tetrahydrouridineand a pharmaceutically acceptable excipient.
 2. A composition for oraladministration comprising about 100 mg decitabine and about 500 mgtetrahydrouridine and a pharmaceutically acceptable excipient.
 3. Thecomposition of claim 1 or 2 for treating a blood disorder in a subject.4. The composition of claim 3, wherein the blood disorder is ahemoglobinopathy or a thalassemia.
 5. The composition of claim 1 or 2for treating a hematological or solid malignancy in a subject.
 6. Thecomposition of claim 5, wherein the malignancy is selected from thegroup consisting of leukemia, lymphoma, multiple myeloma, cancer of thebrain, cancer of the head, cancer of the neck, cancer of the mouth,cancer of the pharynx, cancer of the esophagus, cancer of the stomach,cancer of the intestine, cancer of the thyroid, cancer of the lungs,cancer of the mediastinum, cancer of the thymus, cancer of themesothelium, cancer of the peritoneum, cancer of the bone, cancer of themuscle, cancer of the skin, cancer of the prostate, cancer of thebreasts, cancer of the ovaries, cancer of the uterus, cancer of thevagina, and virally related malignancy.
 7. The composition of any one ofthe preceding claims, wherein the tetrahydrouridine is bio-availableabout 15 to about 180 minutes before the decitabine.
 8. The compositionof claim 7, wherein the tetrahydrouridine is bio-available about 30 toabout 60 minutes before the decitabine.
 9. A composition for oraladministration in the form of a capsule or tablet comprising decitabineand tetrahydrouridine and a pharmaceutically acceptable excipient,wherein the tetrahydrouridine is bio-available about 15 to about 180minutes before the decitabine.
 10. The composition of claim 9, whereinthe tetrahydrouridine is bio-available about 30 to about 60 minutesbefore the decitabine.
 11. The composition of claim 9 or 10, wherein thetetrahydrouridine is located at the surface of the capsule, and thedecitabine is located within the capsule.
 12. A method for treating ablood disorder in a subject, comprising administering to the subject thecomposition of any one of claims 1, 2, and 9-11.
 13. The method of claim12, wherein the blood disorder is a hemoglobinopathy or a thalassemia.14. The method of claim 12, wherein the subject is provided anadditional form of therapy.
 15. The method of claim 12, wherein thesubject is refractory to or intolerant of treatment with hydroxyurea.16. A method for treating a hematological or solid malignancy in asubject, comprising administering to the subject the composition of anyone of claims 1, 2, and 9-11.
 17. The method of claim 16, wherein themalignancy is selected from the group consisting of leukemia, lymphoma,multiple myeloma, cancer of the brain, cancer of the head, cancer of theneck, cancer of the mouth, cancer of the pharynx, cancer of theesophagus, cancer of the stomach, cancer of the intestine, cancer of thethyroid, cancer of the lungs, cancer of the mediastinum, cancer of thethymus, cancer of the mesothelium, cancer of the peritoneum, cancer ofthe bone, cancer of the muscle, cancer of the skin, cancer of theprostate, cancer of the breasts, cancer of the ovaries, cancer of theuterus, cancer of the vagina, and virally related malignancy.
 18. Themethod of claim 16, wherein the subject is provided an additional formof therapy.
 19. A method for decreasing the inter-individual variationin decitabine pharmacokinetics and/or clinical effects in subjects,comprising administering to the subjects the composition of any one ofclaims 1, 2, and 9-11.
 20. A method for extending thetime-above-threshold concentration for depleting DNMT1 with decitabinein a subject and avoiding DNA-damaging high peak levels of decitabine,comprising administering to the subject the composition of any one ofclaims 1, 2, and 9-11.
 21. The method of claim 12 or 16, wherein thesubject is human.
 22. The method of claim 12 or 16, further comprisingobtaining the composition.